Melanoma, one of the most notorious and lethal forms of skin cancer, remains resistant to available treatments. Therefore, novel target-based approaches are needed for the management of this neoplasm. An in-depth knowledge of the genetic controls of cellular proliferation and cell division may provide critical information regarding melanomagenesis. Polo-like kinase 1 (Plk1), a member of highly conserved family of mitotic serine/threonine kinases, plays a critical role in cell division and cycle progression. Plk1 dysregulation has been shown to result in chromosomal instability, aneuploidy and tumor development. However, the functional role of Plk1 in melanoma development is not well understood. In a recent study, we demonstrated that i) Plk1 was over-expressed in both clinical melanoma specimens and cultured human melanoma cells when compared to normal skin and cultured normal melanocytes, respectively; and ii) inhibition of Plk1 resulted in a significant decrease in the viability and growth of melanoma cells. This study suggested that Plk1 may have an important role in melanoma. However, additional studies are needed to assess the functional significance of Plk1 in melanoma and to define its molecular mechanism(s). Our preliminary data have shown that i) Plk1 binds with endogenous Numb (antagonist of Notch and a p53 regulator) both in vitro and in vivo, ii) localization of Plk1 and Numb are reliant upon the expression of each other, and iii) loss of either Plk1 or Numb expression results in a similar cell cycle profile with a G2/M cell cycle arrest. Based on our published study and our preliminary data, we propose to test the hypothesis that Plk1 and Numb work in close concert during mitosis, expression of both are required for proper melanocytic cell division and integrity, and loss of Numb and Plk1 regulation deregulates p53 contributing to increased genomic instability and melanomagenesis. We will challenge this hypothesis in three specific aims. . First, employing retrospective surgical specimens, we will determine the stage-specific association between Plk1, p53 and Numb during melanoma progression. We will employ quantitative AQUA analysis in tissues of different stages during melanomagenesis (common and dysplastic nevi, localized cutaneous melanoma, and melanoma with regional lymph node involvement). Next, in order to define the cooperation between Plk1 and Numb, we will determine the consequence of shRNA mediated knockdown of Plk1 and Numb on one another by assessing the effects on mRNA and protein expression, stability, and localization, as well as mitotic structures in melanoma cells and normal melanocytes. Further, extensive experiments will be conducted to dissect the mechanistic details of the Plk1 and Numb interaction. Finally, we will determine the in vivo relevance of our in vitro findings by determining the consequence of Plk1 and Numb inhibition in athymic nude mice xenografts implanted with melanoma cells with varying Plk1 and Numb expression. Outcome of proposed studies may define the role and cooperation of Plk1and Numb in melanomagenesis. Further, the work proposed in this application is relevant to military Veterans as melanoma incidence is higher in veteran population. Our proposed study may lead to identification of novel strategies for the management of this deadly neoplasm.